Reprogramming of human whartons jelly cells to produce hair cells

ABSTRACT

A method of transforming human cells into mechanosensory hair cells (MHCs), such as inner hear hair cells in the cochlea and vestibular organs, can include: causing human Wharton&#39;s jelly cells (hWJCs) to increase expression of or biological function of HATH1 so as to transform the hWJCs into MHCs. The method can include; administering a nucleic acid that encodes HATH1 to the hWJCs; causing inhibited expression of or biological function of HES1 and/or HES5 in the hWJCs; administering a nucleic acid that inhibits HES1 and/or a nucleic acid that inhibits HES5 to the hWJCs; causing inhibited expression of or biological function of HES1 and/or HES5 in the WJCs by administering a nucleic acid that inhibits HES1 and/or a nucleic acid that inhibits HES5; nucleic acids are administered includes a sequence of SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO: 4.

CROSS-REFERENCE

This patent application claims priority to U.S. Provisional 61/985,075filed Apr. 28, 2014, which provisional application is incorporatedherein by specific reference in its entirety.

GOVERNMENT RIGHTS

This invention was made with government support under R01 AR056347-01awarded by the NIH. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 28, 2015, isnamed K1262.10052US01_SL.txt and is 9,531 bytes in size.

BACKGROUND

Mechanosensory hair cells (MHCs) facilitate hearing and balance in thecochlea and vestibular organs, respectively. MHCs are susceptible todamage from antibiotics, aging, wear and tear, noise-induced stress, anddifferent types of infections. Hearing loss occurs when MHCs aredamaged, and MHCs do not naturally regenerate in humans. Currently,there is no way to artificially regenerate MHCs. Cochlear implants andhearing aides are the only ways to treat hearing loss.

Thus, it would be advantageous to regenerate MHCs in a way to treat orreverse hearing loss or other problems associated with damaged MHCs.

SUMMARY

In one embodiment, a method of transforming human cells intomechanosensory hair cells (MHCs), such as inner hear hair cells in thecochlea and vestibular organs, can include: causing human Wharton'sjelly cells (hWJCs) to increase expression of or biological function ofHATH1 so as to transform the hWJCs into MHCs. In one aspect, the methodcan include administering a nucleic acid that encodes HATH1 to thehWJCs. In one aspect, the method can include causing inhibitedexpression of or biological function of HES1 and/or HES5 in the hWJCs.In one aspect, the method can include administering a nucleic acid thatinhibits HES1 and/or a nucleic acid that inhibits HES5 to the hWJCs. Inone aspect, the method can include causing inhibited expression of orbiological function of HES1 and/or HES5 in the WJCs by administering anucleic acid that inhibits HES1 and/or a nucleic acid that inhibitsHES5. In one aspect, one of more of the nucleic acids are administeredto the WJCs by non-viral nucleic acid delivery. In one aspect, thenon-viral nucleic acid delivery is electroporation. In one aspect, thenucleic acid that encodes HATH1 includes a sequence of SEQ ID NO: 2; thenucleic acid that inhibits HES1 includes a sequence of SEQ ID NO: 3; andthe nucleic acid that inhibits HES5 includes a sequence of SEQ ID NO: 4.

In one embodiment, the method is devoid of administering a nucleic acidthat encodes MATH1.

In one embodiment, the method can include seeding the transforming hWJCsor MHCs onto a substrate shaped as a cochlea and/or vestibular organs.In one aspect, the substrate is a decellularized cochlea and/orvestibular organs. In one aspect, the method can include perfusing thetransforming hWJCs or MHCs onto the decellularized cochlea and/orvestibular organs.

In one aspect, the transforming of the hWJCs to the MHCs occurs within 7days. That is, upon initiation of transformation, such transformationresults in MHCs within 7 days. As such, biomarkers of MHCs can bedetected within 7 days of the initiation of the transformation fromhWJCs.

In one embodiment, a method of providing mechanosensory hair cells(MHCs) to an inner ear of a subject can include: causing human Wharton'sjelly cells (hWJCs) to increase expression of or biological function ofHATH1 so as to transform the hWJCs into MHCs; and implanting the MHCsinto the inner ear of the subject. In one aspect, the implanted MHCs aresufficient to improve hearing and/or balance in the subject. Thegenerated MHCs can be implanted into a cochlea and/or vestibular organsof a subject, such as a subject in need thereof. The subject may havehearing loss or balance problems.

In one aspect, a cell culture system can include: a decellularizedcochlea and/or vestibular organs; and mechanosensory hair cells (MHCs)growing on the decellularized cochlea and/or vestibular organs. In oneaspect, the MHCs are characterized as being derived from human Warton'sjelly cells (WJCs) that have been transfected with a nucleic acid thatencodes HATH1 and a nucleic acid that inhibits HES1 and a nucleic acidthat inhibits HES5. In one aspect, the one or more test substances inthe MHCs include one or more test substances that are not native toWarton's jelly cells (WJCs) or MHCs and that are present in an amount toscreen for biological activity thereof.

In one embodiment, a method of screening compounds for biologicalactivity on mechanosensory hair cells (MHCs) can include: providing thesystem having a decellularized cochlea and/or vestibular organs andmechanosensory hair cells (MHCs) growing on the decellularized cochleaand/or vestibular organs; administering a test substance to the MHCs;and determining whether or not the test substance has biologicalactivity on the MHCs. In one aspect, the test substance is determined tohave a negative biological activity on the MHCs, and the method caninclude: selecting the test substance for further testing of toxicity ofthe test substance on MHCs. In one aspect, the test substance isdetermined to have a positive biological activity on the MHCs, and themethod can include: selecting the test substance for further testing oftherapeutic benefit for treatment of hearing and/or balance loss.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative aspects, embodiments,and features described above, further aspects, embodiments, and featureswill become apparent by reference to the drawings and the followingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing and following information as well as other features ofthis disclosure will become more fully apparent from the followingdescription and appended claims, taken in conjunction with theaccompanying drawings. Understanding that these drawings depict onlyseveral embodiments in accordance with the disclosure and are,therefore, not to be considered limiting of its scope, the disclosurewill be described with additional specificity and detail through use ofthe accompanying drawings.

FIG. 1 includes micrographs of hWJC cells after various treatments withMATH1, HATH1, HES1, and/or HES2.

FIG. 2 includes micrographs of hWJC cultured on cochlear tissue.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented herein. It will be readily understood that the aspects of thepresent disclosure, as generally described herein, and illustrated inthe figures, can be arranged, substituted, combined, separated, anddesigned in a wide variety of different configurations, all of which areexplicitly contemplated herein.

In one embodiment, the present invention includes compositions, systems,and methods for reprogramming of human Wharton's Jelly cells (WJCs) todevelop and behave as mechanosensory hair cells (MHCs), and thereby suchreprogramed WJCs can be generated MHCs. Such generation of MHCs can bethrough different techniques. In one example, genetic manipulation ofthe NOTCH signaling genes in WJCs can cause increased expression forHATH1, and silenced or reduced expression for HES1 and HES5 to form theMHCs. In another example, culturing of WJCs (e.g., hWJCs) in a 3D systemthat mimics the native MHC environment, such as a decellularized cochleaand/or vestibular organs, can form MHCs.

In one aspect, HATH1 can be upregulated in WJCs without down-regulationby HES1 and HES5 to form MHCs. In another aspect, a gene encoding HATH1can be administered to the WJCs along with siRNA or other silencing RNAto down-regulate HES1 and HES5 to form the MHCs. For example, HATH1 canbe over-expressed by delivering HATH1 plasmid DNA to target cells, suchas hWJCs, thus increasing the atonal effect and the potential for MHCdevelopment. At the same time, HES1 and HES5 gene expression can besuppressed by delivering siRNA against HES1 and siRNA against HES5,which can further increase the atonal effect and the potential for MHCdevelopment. In one aspect, the methodology of producing MHCs caninclude delivering HATH1 plasmid DNA to hWJCs using any delivery method,such as viral, non-viral polymers, or any other non-viral method, suchas electroporation or precipitation, where a non-viral delivery methodcan be preferred. In another aspect, the methodology of producing MHCscan include delivering HES1 siRNA and HES5 siRNA to hWJCs using anydelivery method, where a non-viral method can be preferred, such aselectroporation. In one aspect, the methodology for producing MHCs caninclude simultaneously increasing HATH1 expression via non-viraldelivery of HATH1 plasmid DNA and suppressing HES1 and HES5 expressionby the non-viral delivery of siRNA against HES1 and siRNA against HES5.The non-viral delivery of the HATH1 plasmid DNA and HES1 and HES5 siRNAcan be done simultaneously with the same non-viral delivery method.

In one embodiment, the present invention can include using a cochlea,such as a decellularized cochlea and/or vestibular organs, as asubstrate for growing the WJCs into MHCs. Also, the present inventioncan include using a cochlea to investigate the role the physicalarchitecture of the cochlea plays in the development and maintenance ofMHCs in the cochlea, which can be done ex-vivo or in-vitro. Furthermore,the extracellular matrix of the cochlea and/or vestibular organs can beused for a substrate for growing WJCs into MHCs, or using suchextracellular matrix to investigating the role the extracellular matrixplays in the development, maintenance, and repair of MHCs. In oneaspect, the methodology can include seeding WJCs (e.g., hWJCs) on top ofa decellularized cochlea and/or vestibular organs, such as a human, dog,rat, mouse, or other cochlea and/or vestibular organs. The methodologycan include seeding WJCs, such as hWJCs, which have been transfectedwith HATH1 plasmid DNA on top of a decellularized cochlea (e.g., mouse)and/or vestibular organs. Additionally, the methodology can includeseeding WJCs, such as hWJCs, which have been transfected with siRNAagainst HES1 and siRNA against HES5 on top of a decellularized mousecochlea and/or vestibular organs. Accordingly, HATH1 up-regulation canbe induced with or without HES1 and/or HES5 down-regulation, and HES1and/or HES5 down-regulation can be induced with or without HATH1up-regulation. In one aspect, only one of HES1 or HES5 needs to bedown-regulated with or without up-regulation of HATH1. However, in oneaspect, the methodology can include seeding hWJCs that have beentransfected with HATH1 plasmid DNA, siRNA against HES1, and siRNAagainst HES5 on top of a decellularized cochlea (e.g., mouse) and/orvestibular organs.

In one embodiment, the methodology can include perfusing the hWJCs intoa decellularized cochlea and/or vestibular organs. In one aspect, thiscan include perfusing hWJCs that have been transfected with HATH1plasmid DNA into a decellularized cochlea. In one aspect, themethodology can include perfusing hWJCs that have been transfected withsiRNA against HES1 and siRNA against HES5 into a decellularized cochlea.In one aspect, the methodology can include perfusing hWJCs that havebeen transfected with HATH1 plasmid DNA, siRNA against HES1, and siRNAagainst HES5 into a decellularized cochlea. While decellularized mousecochlea has been used and described, any mammal decellularized cochleacan be used.

Culturing the WJCs in a decellularized cochlea provides severaladvantages. For example, the WJCs can be cultured in a 3D environmentthat physically and mechanically mimics the environment in which MHCsdevelop. The WJCs seeded on cochleae can be studied in real-time outsideof the body. The use of cochleae as scaffolds provides a culturingsystem that is both 3D and flexible in design. Different cell types orcells with different treatments can be seeded in combination orindividually into the cochlea, enabling the study of a more advancedsystem for MHC and sensory epithelium development. Also, using thecochlea shape as a 3D culture system provides a template in the study ofMHC development and regeneration. A 3D culture system provides a meansto examine how signaling pathways change when cells are cultured in anenvironment native to MHC and sensory epithelium instead of in 2D. Thiscan also be done with vestibular organs.

In one aspect, the 3D culture system may be used to screen for potentialototoxic agents and therapeutic agents, which could accelerate theearly-stage development of new treatments for damaged sensoryepithelium. As such, hWJCs can be grown on a cochlea-shaped substrateand/or vestibular organs, and then treated to induce formation of MHCwith or without test compounds to determine whether they promote orinhibit formation of the MHCs. Known screening methods can be performedto find hits for further study for effects in MHCs generation.

In one embodiment, the hWJCs can be manipulated to function similar tomesenchymal stem cells, and can be used in MHC regeneration. In apreferred manipulation, the process can include simultaneouslyincreasing HATH1 expression while suppressing HES1 and HES5 expression.The extracellular matrix and architecture of the cochlea are widelythought not to be the primary components that affect MHC differentiationand maintenance. Thus, utilization of the extracellular matrix as anextracellular material for guiding stem cells toward a MHC phenotype asdescribed herein is surprising and unexpected.

The hWJCs are similar to bone marrow stem cells, which means hWJCs aremultipotent stem cells that can differentiate into any tissueoriginating from the mesoderm germ layer (e.g., cartilage, bone, fat,muscle, etc.). Thus, hWJCs are ideal for applications that focus oncartilage, bone, fat, or muscle regeneration. It was previously thoughtthat hWJCs may not be able to differentiate any other tissue types thatare not connective tissue or found in the mesoderm germ layer. The hWJCswere previously thought to not be able to differentiate down any othernon-mesoderm lineages, and thus were previously considered to be notideal for neuron applications or intestinal applications. HATH1 deliveryto hWJCs to transform them to MHCs was surprising and unexpected.

When a cochlea tissue is decellularized, all that is left is theextracellular matrix with no biochemical signals. Thus, a decellularizedcochlea tissue is an inert biomaterial to grow cells on. However, thecochlea has a very unique architecture, and the architecture andphysical properties can play a role in development of MHCs. Thus, thehWJCs exhibited phenotypic markers exclusive to MHCs after being seededon or perfused inside the decellularized cochlea. This can be the samefor vestibular organs.

The transcription factor atonal homolog 1 (ATOH1) has multiplehomologues that are functionally conserved between species and isresponsible for the generation of sensory hair cells. To evaluatepotential functional differences between homologues, human and mouseatoh1 were delivered to human umbilical cord mesenchymal stromal cellsfrom Wharton's jelly. Delivery of the human atonal homolog, HATH1, tohuman Wharton's jelly cells demonstrated superior expression of innerear hair cell markers and characteristics compared to delivery of themouse homolog, MATH1 Inhibition of HES1 and HES5 signaling furtherincreased the potency of the atonal effect. Transfection of Wharton'sjelly cells with HATH1 DNA, HES1 siRNA, and HES5 siRNA displayedpositive identification of key hair cell and support cell markers foundin the organ of Corti. In the first side-by-side evaluation of HATH1 andMATH1 in human cells, substantial differences were observed, suggestingthat the two atonal homologues may not be interchangeable betweenspecies.

In one embodiment, by over-expressing a target gene (e.g., HATH1) forMHC differentiation in human umbilical cord mesenchymal stem cells(hUCMSCs)) also known as human Wharton's jelly cells (hWJCs), whileusing siRNA to block negative regulators (HES1 and HES5) of the targetgene can generate neuronal-like cells similar to the vestibular-cochlearnerve. The cells can be referenced as as human Wharton's Jelly Cells(hWJCs), and referred to as human umbilical cord mesenchymal stem cells(hUCMSCs). After treating hUCMSCs with HATH1 pDNA and HES1 and HES5siRNA, there were differences between treated hUCMSCs and untreatedhUCMSCs. Over the course of 10 days, bright-field microscopy revealeddistinct differences in the morphological character of cells from bothgroups. Untreated hUCMSCs remained spindle-like in shape over the courseof 10 days. Treated hUCMSCs became elongated and developed synaptic-likeprocesses over the course of 10 days. Four days after treatment, treatedhUCMSCs displayed bipolar elongation. Membrane staining revealed thatdistinct populations of cells were immerging 24 hours after treatment.Treated hUCMSCs take up the dye much more strongly than untreatedhUCMSCs. The membrane stain is a method used to identify actively firingneurons and cells with high metabolic activity at the cell membrane.Over the course of 10 days, treated hUCMSCs continued to maintain apopulation with significantly increased membrane dye uptake as incomparison to the untreated hUCMSCs. Immunocytochemistry revealed theactive protein expression of ATOH1 in treated hUCMSCs 24 hours aftertreatment, whereas untreated hUCMSCs did not reveal any ATOH1 proteinexpression. 10 Days after treatment, cell characterization via flowcytometry revealed that untreated hUCMSCs had maintained stem cellcharacter, whereas treated hUCMSCs had started to shift away fromestablished stem cell characterization parameters. Interestingly, bothcell characterization via flow cytometry and gene expression via RT-qPCRrevealed that while treated hUCMSCs moved away from the original stemcell phenotype, some of the treated hUCMSCs maintained high expressionof a key pluripotent marker after treatment. Thus, based on the datahUCMSCs are responsive to treatment with HATH1 pDNA and HES1 and HES5siRNA, and that such treatment has moved hUCMSCs toward an inner eartissue phenotype. The treated hUCMSCs move toward what appears to beneuronal phenotype similar in character to the vestibular-cochlear nervethat transmits sensory information from IEHCs to the brain regardingbalance and hearing.

In one aspect, HATH1 plasmid DNA delivered in combination with HES1 andHES5 siRNA to hUCMSCs can produce neuron-like cells. Using the plasmidDNA/siRNA combination for nucleic acid delivery has enabled creation ofneuron-like cells efficiently and robustly. Thus, these neuron-like celltypes may be used for drug screen testing, disease modeling, as tissuetransplants, or any other biotechnological use.

In one embodiment, the methodology of generating MHCs can be used fortreating sensorineural hearing loss, such as in mammals. The generatedMHCs can be used to regenerate hair cells in the inner ear. Thus, thegenerated MHCs are able to renew and regenerate to repair damage. Thegenerated MHCs can be obtained and then implanted in a subject. Thegenerated MHCs can be obtained with or without the cochlea and/orvestibular organ substrates, and can be implanted with or without thecochlea and/or vestibular organ substrates. The implantation can be fortherapies to treat or restore hearing loss or balance loss.

In one aspect, the generated MHCs can be implanted into inner earepithelium, such as at the organ of Corti in the cochlea, and/or in theutricle and/or saccule in the vestibular organs. The generated MHCs canbe implanted with or without the growth substrate. This can provideregenerated MHCs in precise quantities, types, and spacing of both haircells (e.g., regenerated MHCs) and supporting cells to enable properreception and transmission of neurosensory signals. This can be used fortreatment of hearing loss or balance.

In one aspect, the generated MHCs can be used to partially or fullyrestore or repair inner ear sensory epithelium to a condition that isfunctionally similar or equivalent its original state. The methodologycan be for generation of new hair cells without over-generation of haircells. This can be used as to control the amount, density anddistribution of the hair cells on the substrate. The generated MHCs canhelp improve hearing and/or balance after damage or have various otheruses.

In one embodiment, ex-vivo or in-vitro generation of MHCs and thenimplantation, can overcome problems of using genetic manipulation invivo that can result in overexpression of ATOH1 in supporting cells.Such overexpression of ATOH1 in supporting cells can unfavorably resultin direct transdifferentiation, not regeneration. As such, thedevelopment of a MHC model outside the body may have a significantimpact on further understanding how hair cells develop and are damaged,which may lead to new approaches for developing a therapy or a model forcompound screening, such as to determine ototoxic drugs or otherbioactive agents. The MHC model can be only generated MHCs or generatedMHCs on decellularized cochlea and/or vestibular organs. Such MHC modelcan be studied in the presence of compounds (e.g., test substances) todetermine if such compounds are ototoxic or have other bioactiveproperties. Also, the MHC model can be studied in the presence ofcompounds to determine if such compounds promote further improvement orinhibition of the MHCs.

In one embodiment, HATH1 transfected into hWJCs can result in cells thatexpress critical markers associated with hair cells (e.g., MHCs) andneural epithelium. It is noted that MATH1 and HATH1 gene delivery inhuman cells has now been demonstrated that HATH1 expression differedfrom MATH1 expression in cells from human tissue, and HATH1 expressionis superior for generating MHCs. In hWJCs, treatment with HATH1displayed significant immediate increases in mRNA and protein expressionof key hair cell markers, as compared to cells treated with MATH1, whichdisplayed limited increases in gene expression and protein expression 1day after transfection. The positive identity of GFAP in cellsco-transfected with HATH1 and siRNA against HES1 and HES5 suggested aninitial differentiation toward a neural-like phenotype. Myosin VIIaexpression is expected if cells are differentiating toward a hair celllineage, but HES5 expression is surprising, because HES5 encouragessupport cell differentiation by negatively regulating ATOH1, and therebythe results are surprising and unexpected. The positive expression ofHES5 both at the gene and protein level suggested that hWJCs may bedifferentiating into both hair cells and supporting cells concurrently.The significant up-regulations GFI1 gene expression in cellsco-transfected with HATH1 and siRNA against HES1 and HES5 suggest thatpresentation of a hair cell phenotype had started within at least asub-population of treated cells. The gene expression and proteinexpression findings combined with the visual morphological changes inHATH1-transfected cells implied that some level of neuronaldifferentiation had taken place outside of the body, with limitedstimulation by HATH1 and intercellular mediators of the NOTCH pathway.Accordingly, the presumption that atonal homologues are interchangeablebetween species is incorrect. This may be the case because the currentstudy found that hWJCs showed increased gene, protein, and morphologicalfeatures as well as increased viability when transfected with HATH1. Inaddition, transduction channels characteristic of hair cells and activeneurons were implicated in the cell membranes of HATH1-transfected cellsand cells co-transfected with HATH1 and siRNA against HES1 and HES5based on the superior infiltration dye into hWJCs.

In one aspect, culturing treated cells in a three-dimensionalenvironment similar to the native cochlea or co-culture of treated cellswith a combination of native hair cells and support cells may furtherenhance the atonal effect and potential display of hair cellcharacteristics. The culturing can produce cells or tissues that can beimplanted and may also be used for various treatments that can beimproved by growing the MHCs in the natural locations.

In summary, the data revealed that hWJCs transfected with HATH1displayed far superior expression of key hair cell markers in relationto presentation of mRNA transcripts, proteins, and morphologicalfeatures in contrast to hWJCs transfected with MATH1. The developmentand presentation of hair cell markers were further enhanced whenHATH1-transfected hWJCs were co-transfected with siRNA against HES1 andHES5. The current study demonstrated that hWJCs can be manipulatedoutside of a target tissue to produce a rare and complex phenotype thatmay aid in illuminating how hair cells develop in the human body.

In one embodiment, a method of increasing expression of inner ear haircell markers in human cells can include: administering human atonalhomolog HATH1 to human Wharton's jelly cells (hWJCs) so as to increaseexpression of inner ear hair cell markers in the hWJCs. In one aspect,the increased expression is compared to when administering mouse homologMATH1 to hWJCs. In one aspect, atonal homolog HATH1 is administered in aplasmid and transfected into the hWJCs. Such a method can also includeinhibiting HES1 and/or HES5 signaling. In one aspect, the method caninclude administering HES1 siRNA and/or HES5 siRNA to the hWJCs. In oneaspect, the method can include transfection of the hWJCs with HATH1 DNA,HES1 siRNA, and HES5 siRNA together. In one aspect, confirmation of themethod being useful as described herein can include identification ofhair cell and support cell markers found in organ of Corti.

In one embodiment, a method of inducing differentiation of umbilicalcord stem cells to inner ear hair cells can include: administering humanatonal homolog HATH1 to hWJCs so as to increase expression of inner earhair cell markers in the hWJCs. In one aspect, the increased expressionis compared to when administering mouse homolog MATH1 to hWJCs. In oneaspect, atonal homolog hath1 is administered in a plasmid andtransfected into the Wharton's jelly cells. In one aspect, the methodcan include inhibiting HES1 and HES5 signaling. In one aspect, themethod can include administering HES1 siRNA and HES5 siRNA to the hWJCs.In one aspect, the method can include transfection of hWJCs with HATH1DNA, HES1 siRNA, and HES5 siRNA. In one aspect, confirmation of themethod being useful as described herein can include identification ofhair cell and support cell markers found in organ of Corti.

EXPERIMENTAL

Human Wharton's jelly cells (hWJCs) were isolated from Wharton's jellyof five human umbilical cords.

Two PrecisionShuttle mammalian vectors with independent turboGFPexpression from OriGene (Rockville, Md.) were used to deliver targetgenes to hWJCs. Cloning and verification services were provided by BlueHeron (Blue Heron Biotech LLC, Bothell, Wash.) to manufacture thevectors. In one vector, a MATH1 insert (NCBI GenBank ID: NM-007500.4,the sequence thereof incorporated herein by specific reference, SEQ IDNO: 1) was cloned in, and in the other vector a HATH1 insert (NCBIGenBank ID: U61148.1, the sequence thereof incorporated herein byspecific reference, SEQ ID NO: 2) was cloned in. The MATH1 and HATH1gene inserts were driven by a cytomegalovirus (CMV) promoter followed bya Kozak sequence, and the turboGFP gene was driven by a Simian virus 40(SV40) promoter. The PrecisionShuttle vectors contained a Kanamycinresistance gene for bacterial selection.

Based on data (data not shown) custom HES1 siRNA (Hs_hes1_(—)5, GeneAccession no.: NM_(—)005524, Gene ID: 3280, the sequence thereofincorporated herein by specific reference, SEQ ID NO: 3) modified with3′-Alexa Fluor 555 and custom HES5 siRNA (Hs_hes5_(—)5, Gene Accessionno.: NM_(—)001010926, Gene ID: 388585, the sequence thereof incorporatedherein by specific reference, SEQ ID NO: 4) modified with 3′-Alexa Fluor647 (Qiagen) were selected for experiments.

All cells were suspended at a concentration of 5×10⁵ cells per 100-4,solution at one of the five following ratios: 100 μL 4DNP1 (Untreated),95 μL 4DNP1: 5 μL MATH1 pDNA (1 μg per μL) (MATH1), 95 μL 4DNP1: 5 μLHATH1 pDNA (1 μg per μL) (HATH1), 99 μL 4DNP1: 0.5 μL HES1 siRNA (100nM): 0.5 μL HES5 siRNA (100 nM) (H1/H5), 94 μL 4DNP1: 5 μL MATH1 pDNA (1μg per μL): 0.5 μL HES1 siRNA (100 nM): 0.5 μL HES5 siRNA (100 nM)(MATH1/H1/H5), 94 μL 4DNP1: 5 μL HATH1 pDNA (1 μg per μL): 0.5 μL HES1siRNA (100 nM): 0.5 μL HES5 siRNA (100 nM) (HATH1/H1/H5). The untreatedcontrol cells were not nucleofected and were immediately pipetted into6-well plates (BD Biosciences) or Nunc™ Lab-Tek™ 8-well chamberedcoverglass slides (Thermo Scientific, Waltham, Mass.) pre-coated withFibronectin (BD Biosciences) containing 1.5 mL or 0.5 mL, respectively,of 37° C. pre-warmed traditional hWJC medium. Cells were transfected ina 4D Nucleofector™ (Lonza) and nucleofected with the FF-104 program.

At 1, 3, and 7 days after transfection, cells were collected andharvested for gene expression analysis via real time quantitativepolymerase chain reaction (RT-qPCR), and RNA was collected from eachcell sample. RNA was converted to cDNA. Three replicates from each offive umbilical cords (n=5) were taken for gene expression analysis at 1,3, and 7 days post transfection. Live cell fluorescent imaging and flowcytometry was performed. The hWJCs were collected for live stain imaging24 hours after transfection. Cells from each cord were stained andimaged 7 days after transfection under a confocal microscope. At 1, 3,and 7 days after transfection, cells were collected forimmunocytochemistry.

Primary antibodies were pre-conjugated to Quantum Dots (Qdot®) Thefollowing primary antibodies were conjugated to the following Qdots:Anti-human ATOH1 (Millipore, Billerica, Mass.) pre-conjugated to Qdot525 (1:200; Life Technologies), Anti-human HES1 (Millipore)pre-conjugated to Qdot 565 (1:200; Life Technologies), Anti-human MYOSINVIIa (Novus, Littleton, Colo.) pre-conjugated to Qdot 605 (1:500, LifeTechnologies), Anti-human HES5 (Millipore) pre-conjugated to Qdot 655(1:200, Life Technologies), and Anti-human GFAP (Millipore)pre-conjugated to Qdot 800 (1:100, Life Technologies). Cells were imagedusing confocal microscopy. Cells were collected from each umbilical cordat 1, 3, and 7 days after transfection and images were taken fromindividual wells on 8-well chambered glass slides.

Analysis of stem cell characteristics was performed. A sub-culture ofcells from each cord was characterized through cell surface markeridentification via flow cytometry. Primary cell surface antibodies andsecondary antibodies were added sequentially one-at-a-time perincubation-wash cycle to avoid cross-reaction; however, pre-conjugatedprimary antibodies with secondary antibodies were added simultaneously.A single incubation-wash cycle consisted of adding a primary antibody,secondary fluorescent antibody, or primary antibody pre-conjugated to aspecific fluorescent secondary antibody to the cell suspension. Cellsurface marker antibodies and secondary antibodies were added in thefollowing order at the following ratios: STRO-1 Mouse IgM (2.5:200) (1mg per mL; R&D Systems, Minneapolis, Minn.); Alexa Fluor 568® RabbitAnti-Mouse IgG (2:200) (2 mg per mL; Life Technologies); CD105 Mouse IgG(2.5:200) (1 mg per mL; R&D Systems); Qdot® 525 donkey anti-mouse IgG(2:200) (1 μM; Life Technologies); Human CD45 pre-conjugated to Qdot®800 (2:200) (Life Technologies); Human CD73 pre-conjugated to FITC(5:200) (BD Biosciences); Human CD34 pre-conjugated to Brilliant Violet(5:200) (BD Biosciences); Human CD90 pre-conjugated to APC (5:200) (BDBiosciences). hWJCs were analyzed by flow cytometry on a MoFlo XDF FACS(Beckman Coulter). Positive identification of cell markers was definedas fluorescent emission that exceeded the fluorescent threshold of cellsstained with corresponding isotype (negative) controls.

Cells transfected with HATH1 showed greater cell density than cellstransfected with MATH1. hWJCs were transfected via Nucleofection™, anelectroporative technique (Lonza, Basel Switzerland), with one of fivedifferent treatments: MATH1 pDNA, HATH1 pDNA, siRNA against HES1 andHES5, MATH1 pDNA and siRNA against HES1 and HES5, or HATH1 pDNA andsiRNA against HES1 and HES5. At 24 hours post-transfection, there was anoticeable visual difference in cell numbers between cells treated withHATH1 versus cells treated with MATH1 (see Table 1). Twenty-four hoursafter transfection, flow cytometry revealed that there were 1.9 timesmore viable cells transfected with HATH1, and 2.2 times more viablecells transfected with HATH1 and siRNA against HES1 and HES5, thanviable cells transfected with MATH1 (Table 1). In addition, 24 hourspost-transfection cells co-transfected with HATH1 and siRNA against HES1and HES5 displayed 3.7 times more viable cells than cells co-transfectedwith MATH1 and siRNA against HES1 and HES5. At 24 hourspost-transfection, cells transfected with HATH1 displayed transfectionefficiency that was 0.2 times greater than cells transfected with MATH1.Moreover, 7 days after transfection, cell counts revealed that therewere 2.8 times more viable cells transfected with HATH1, and 3.1 timesmore viable cells transfected with HATH1 and siRNA against HES1 andHES5, than cells transfected with MATH1. At 7 days post-transfectioncells co-transfected with HATH1 and siRNA against HES1 and hes5displayed 2.8 times more viable cells than cells co-transfected withMATH1 and siRNA against HES1 and HES5.

Only cells transfected with HATH1 revealed significant visual changes inmorphology. Visual morphological differences were evident betweenuntreated control cells and cells treated with HATH1 and siRNA againstHES1 and HES5 starting at Day 3 (see incorporated provisionalapplication). Cells treated with MATH1 displayed a fibroblasticmorphology, consistent with hWJCs. However, cells treated with HATH1,siRNA against HES1 and HES5, or a combination of both showed anelongated cell body with small projections expanding away from the cellbody. Cells treated with both HATH1 and siRNA against HES1 and HES5displayed a bipolar phenotype with cell extensions reaching out from thenucleus and terminating with multiple slender projections,uncharacteristic of hWJCs.

HATH1-transfected cells revealed infiltration of lipophilic dye. Tofurther evaluate the development of morphological features of haircells, controls and treatment groups were stained 7 days aftertransfection with a lipophilic dye that is known to enter cells throughtransduction channels found in hair cells and neurons. Cells treatedwith HATH1 stained positive for dye, as did cells treated with onlysiRNA against HES1 and HES5. Dye entered HATH1-transfected cells morereadily and robustly than MATH1-transfected cells. Across cells from allfive umbilical cords, we saw positive staining in the greatestquantities in cells treated with HATH1 only or HATH1 and HES1 siRNA andHES5 siRNA. The amount of positive staining varied between cells treatedonly with HATH1 and cells treated with HATH1 and siRNA against HES1 andHES5 (Table 2). Limited infiltration of dye was observed in some of thesamples co-transfected with math1 and siRNA against HES1 and HES5 acrosscell samples from all five umbilical cords.

HATH1-transfected cells up-regulated different genes fromMATH1-transfected cells. Gene expression was evaluated across alltreated cells from all human umbilical cords at 1, 3, and 7 days aftertransfection. The common trend observed across all analyzed genes was anup-regulation of gene expression 1 day after transfection, and geneexpression levels returned to levels similar to untreated controls 7days after transfection (see incorporated provisional application). Geneexpression in MATH1-transfected cells did not significantly differ fromuntreated control cells within the 7-day time period followingtransfection, except for JAGGED2, HES1, and HES5 genes, 1 day aftertransfection. Cells co-transfected with MATH1 and siRNA against HES1 andHES5 displayed no significant gene expression differences from untreatedcontrol samples within the 7-day time period following transfection.MATH1-transfected cells failed to show any significant increase in geneexpression over the 7-day time period, whereas HATH1-transfected cellsshowed significant (p<0.05) increases in gene expression 1 day aftertransfection compared to untreated control cells in ATOH1 (4.5×10⁵ foldchange), HES1 (6.8 fold change), HES5 (33.3 fold change), and MYOSINVIIA (6.5 fold change). Cells that were co-transfected with HATH1 andsiRNA against HES1 and HES5 displayed significant (p<0.05) increases ingene expression across ATOH1 (3.2×10⁵ fold change), HES5 (17.6 foldchange), MYOSIN VIIa (11.0 fold change), GFI1 (2.9 fold change), andJAGGED2 (2.4 fold change) 1 day after transfection. HWJCs co-transfectedwith HATH1 and siRNA against HES1 and HES5 displayed significant(p<0.05) increases in gene expression across myosin VIIa (9.1 foldchange) and JAGGED2 (1.2 fold change) 3 days after transfection.

Only Cells Transfected with HATH1 Displayed Prolonged Protein Expressionof Myosin VIIa.

Cells were analyzed for protein expression via immunocytochemistry 1 dayand 7 days after transfection (Table 3). All treated cells displayedpositive identification of MYOSIN VIIa and HES5 1 day aftertransfection. However, cells co-transfected with HATH1 and siRNA againstHES1 and HES5 displayed positive identification of glial fibrillaryacidic protein (GFAP) 1 day after transfection. MATH1-transfected cellsdisplayed a visual decrease in myosin VIIa and HES5 expression, whereasHATH1-transfected cells displayed a visual increase in immunostainingfor myosin VIIa and HES5 7 days after transfection. No GFAP expressionwas detected in any treated group 7 days after transfection. Untreatedcontrol cells displayed no presentation of any hair cell marker proteinsat 1 day or 7 days after culture.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptorwas stained to identify active calcium permeable ion channels.MATH1-transfected cells and cells co-transfected with MATH1 and siRNAagainst HES1 and HES5 visually displayed limited presentation of theAMPA receptor, whereas HATH1-transfected cells, and cells co-transfectedwith HATH1 and siRNA against HES1 and HES5 displayed strong presentationof active AMPA receptors. Only minor changes in cell surface markersobserved between untreated and treated cells.

The hWJCs were characterized for CD markers associated with stem cells10 day after transfection and found no significant changes betweenuntreated and treated cells. All cell populations were strongly negativefor CD34 and CD45, which indicated that cell populations werenon-hematopoietic. Additionally, all cell populations displayedpresentation of CD73, CD90, and CD105, which are surface markers foundon mesenchymal stem cells.

FIG. 1 shows that untreated hWJCs displayed a fibroblast-like morphology4 days after plating. MATH-1-transfected hWJCs mostly displayed afibroblastic morphology. The hWJCs transfected with HATH1 and/or siRNAagainst HES1 and HES5 displayed an elongated bipolar morphology 4 daysafter transfection, and multiple cell morphologies were observed 6 daysafter transfection, which included elongated bipolar morphologies,bulb-like morphologies, branching morphologies and select cells appearedto be “pear-shaped”. Cell density steadily increased after transfection.Images were obtained using plastic differential interference contrastmicroscopy (PlasDIC) (Zeiss). Images are representative of cells fromfive different umbilical cords (n=5). MATH-1 represents cellstransfected with MATH-1 pDNA. HATH1 represents cells transfected withHATH1 pDNA. H1/H5 represents cells transfected with HES1 and HES5 siRNA.MATH-1/H1/H5 represents cells co-transfected with MATH-1 pDNA, HES1siRNA, and HES5 siRNA. HATH1/H1/H5 represents cells co-transfected withHATH1 pDNA, HES1 siRNA, and HES5 siRNA. Scale Bar=50 μm.

FIG. 2 shows that even in the absence of any gene delivery,decellularized cochleae alone can induce expression of MYO7A and SOX2 inhWJCs within only 4 days. Untreated hWJCs cultured on fibronectin-coatedglass showed no expression of MYO7A or SOX2 (Top Row). However, whenuntreated hWJCs were cultured in cochlear tissue from deceased C57BL/6mice (Row 2), MYO7A was positively identified as well as SOX2. Thedecellularized cochlea also enhanced the differentiation ofHATH1-transfected hWJCs toward the hair cell phenotype at 4 days.HATH1-transfected hWJCs grown on fibronectin-coated glass displayedpositive presentation of MYO7A and SOX2 (Row 3). Remarkably,HATH1-transfected hWJCs showed an intense presentation of MYO7A andlittle SOX2 presentation when cultured in cochlear tissue (Row 4). Cellnuclei were stained with Ethidium Bromide (Blue). Cells wereimmunostained for MYO7A (Green), a motor protein found on stereocilia inhair cells, and SOX2 (Red), a marker for neuronal lineage commitmentusually up-regulated before hair cell differentiation. MYO7A=myosinVIIA, scale bar=20 μm.

The cochlea substrate for seeding with cells can decellularized. Suchdecellularization can include: dissection of mouse carcass and removalof bulba; isolation of inner ear organs and soak the same withantibiotic solution; decellularize the organs with 1% SDS; decalcifywith 10% EDTA. This process results in decellularized cochlea that canbe seeded with WJCs that can be transformed into MHCs using theprocesses described herein.

Both untreated hWJCs and transfected hWJCs displayed varyingpresentations of MHC and neuron markers after only seven days of culturewithin decellularizied mouse cochleae.

One skilled in the art will appreciate that, for this and otherprocesses and methods disclosed herein, the functions performed in theprocesses and methods may be implemented in differing order.Furthermore, the outlined steps and operations are only provided asexamples, and some of the steps and operations may be optional, combinedinto fewer steps and operations, or expanded into additional steps andoperations without detracting from the essence of the disclosedembodiments.

The present disclosure is not to be limited in terms of the particularembodiments described in this application, which are intended asillustrations of various aspects. Many modifications and variations canbe made without departing from its spirit and scope, as will be apparentto those skilled in the art. Functionally equivalent methods andapparatuses within the scope of the disclosure, in addition to thoseenumerated herein, will be apparent to those skilled in the art from theforegoing descriptions. Such modifications and variations are intendedto fall within the scope of the appended claims. The present disclosureis to be limited only by the terms of the appended claims, along withthe full scope of equivalents to which such claims are entitled. It isto be understood that this disclosure is not limited to particularmethods, reagents, compounds compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” and the like include the number recited andrefer to ranges which can be subsequently broken down into subranges asdiscussed above. Finally, as will be understood by one skilled in theart, a range includes each individual member. Thus, for example, a grouphaving 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, agroup having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells,and so forth.

From the foregoing, it will be appreciated that various embodiments ofthe present disclosure have been described herein for purposes ofillustration, and that various modifications may be made withoutdeparting from the scope and spirit of the present disclosure.Accordingly, the various embodiments disclosed herein are not intendedto be limiting, with the true scope and spirit being indicated by thefollowing claims. All references recited herein are incorporated hereinby specific reference in their entirety.

Tables

TABLE 1 Cell Viability Groups Time MATH1/siHES1/ HATH1/siHES1/ PointUntreated MATH1 HATH1 siHES1/siHES5 siHES5 siHES5 Day 1 86.7 ± 5.6%  57± 49% 49 ± 30% 72 ± 13% 32 ± 41% 49 ± 29% Day 3 78 ± 36% 55 ± 16% 60 ±15% 80.2 ± 7.2%  44 ± 18% 63 ± 16% Day 7 66 ± 13% 69 ± 19% 66 ± 11% 80 ±12% 52 ± 20% 68 ± 27% Percentages represented as the total number ofviable cells out of entire population assessed. Values are representedas mean with standard deviation taken from cell populations from 5different donors (n = 5)

TABLE 2 Identification of Transduction Channels via Lipophilic FM ® 1-43Staining Groups Donor MATH1/siHES1/ HATH1/siHES1/ Population UntreatedMATH1 HATH1 siHES1/siHES5 siHES5 siHES5 A − − + + − ++ B − − ++ + − + C− − + + − ++ D − − ++ + − ++ E − − ++ + − + (−) = Negative detection ofFM ® 1-43 Staining; (+) = positive detection FM ® 1-43 Staining; (++)strong positive detection of FM ® 1-43 staining.

TABLE 3 Identification of Hair Cell Marker MYO7A Groups DonorMATH1/siHES1/ HATH1/siHES1/ Population Untreated MATH1 HATH1siHES1/siHES5 siHES5 siHES5 Day 1 − + + + + + Day 7 − − ++ − + ++ (−) =Negative presentation of MYO7A; (+) = positive presentation MYO7A; (++)strong positive presentation of MYO7A.

TABLE 4 Presentation of Hair Cell and Neuron Markers 7 DaysPost-Transfection Groups DC- Untreated DC- DC- DC- DC- Markers DC hWJCsGFP HATH1 siHES1/siHES5 HATH1/siHES1/siHES5 Hair Actin − ++ ++ ++ + +Cell MYO7A − ++ + ++ + ++ Neuron NEUROG1 − + + ++ + + (−) = Negativepresentation; (+) = positive presentation; (++) strong positivepresentation. DC = Decellularized Cochlea; hWJCs = human Wharton's jellycells; GFP = Green Fluorescent Protein; HATH1 = Cells transfected withHATH1; siHES1 = siRNA against HES1; siHES5 = siRNA against HES5.

1. A method of transforming human cells into mechanosensory hair cells(MHCs), the method comprising: causing human Wharton's jelly cells(hWJCs) to increase expression of or biological function of HATH1 so asto transform the hWJCs into MHCs.
 2. The method of claim 1, comprisingadministering a nucleic acid that encodes HATH1 to the hWJCs.
 3. Themethod of claim 1, comprising: causing inhibited expression of orbiological function of HES1 and/or HES5 in the hWJCs.
 4. The method ofclaim 3, comprising administering a nucleic acid that inhibits HES1and/or a nucleic acid that inhibits HES5 to the hWJCs.
 5. The method ofclaim 2, comprising: causing inhibited expression of or biologicalfunction of HES1 and/or HES5 in the WJCs by administering a nucleic acidthat inhibits HES1 and/or a nucleic acid that inhibits HES5.
 6. Themethod of claim 5, wherein the nucleic acids are administered to theWJCs by non-viral nucleic acid delivery.
 7. The method of claim 6,wherein the non-viral nucleic acid delivery is electroporation.
 8. Themethod of claim 5, wherein: the nucleic acid that encodes HATH1 includesa sequence of SEQ ID NO: 2; the nucleic acid that inhibits HES1 includesa sequence of SEQ ID NO: 3; and/or the nucleic acid that inhibits HES5includes a sequence of SEQ ID NO:
 4. 9. The method of claim 1,comprising: seeding the transforming hWJCs or MHCs onto a substrateshaped as a cochlea.
 10. The method of claim 9, wherein the substrate isa decellularized cochlea.
 11. The method of claim 10, comprisingperfusing the transforming hWJCs or MHCs onto the decellularizedcochlea.
 12. The method of claim 1, comprising transforming the WJCs tothe MHCs within 7 days.
 13. A method of providing mechanosensory haircells (MHCs) to an inner ear of a subject, the method comprising:causing human Wharton's jelly cells (hWJCs) to increase expression of orbiological function of HATH1 so as to transform the hWJCs into MHCs; andimplanting the MHCs into the inner ear of the subject.
 14. The method ofclaim 13, wherein the implanted MHCs are sufficient to improve hearingand/or balance in the subject.
 15. A cell culture system comprising: adecellularized cochlea; and mechanosensory hair cells (MHCs) growing onthe decellularized cochlea.
 16. The cell culture system of claim 15,wherein the MHCs are characterized as being derived from human Warton'sjelly cells (WJCs) that have been transfected with a nucleic acid thatencodes HATH1 and a nucleic acid that inhibits HES1 and a nucleic acidthat inhibits HES5.
 17. The cell culture system of claim 15, comprisingone or more test substances in the MHCs, the one or more test substancesare not native to Warton's jelly cells (WJCs) or MHCs and are present inan amount to screen for biological activity thereof.
 18. A method ofscreening compounds for biological activity on mechanosensory hair cells(MHCs), the method comprising: providing the system of claim 15;administering a test substance to the MHCs; and determining whether ornot the test substance has biological activity on the MHCs.
 19. Themethod of claim 18, wherein when the test substance is determined tohave a negative biological activity on the MHCs, the method comprising:selecting the test substance for further testing of toxicity of the testsubstance on MHCs.
 20. The method of claim 18, wherein the testsubstance is determined to have a positive biological activity on theMHCs, the method comprising: selecting the test substance for furthertesting of therapeutic benefit for treatment of hearing and/or balanceloss.